1. Field of the Invention
The present invention relates to in vivo expression technology, and more particularly to a method for selecting microbial virulence genes that are specifically induced in host tissues.
2. Description of the State of the Art
An infection of the human body by a pathogen, or disease-producing microorganism, results in disease when the potential of the microorganism to disrupt normal bodily functions is fully expressed. Some disease-producing microorganisms possess properties, referred to as virulence factors, that enhance their pathogenicity and allow them to invade host or human tissues and disrupt normal bodily functions. The virulence of pathogens, that is, their ability to induce human disease, depends in large part on two properties of the pathogen, invasiveness and toxigenicity. Invasiveness refers to the ability of the pathogen to invade host or human tissues, attach to cells, and multiply within the cell or tissues of the human body. Toxigenicity refers to the ability of a pathogen to produce biochemicals, known as toxins, that disrupt the normal functions of cells or are generally destructive to cells and tissues.
Scientists can develop better therapeutic and diagnostic approaches against pathogenic microbes if they understand better the molecular mechanisms of the specific pathogenic microbes or microorganisms that allow them to circumvent the host's, e.g., human body, immune system and initiate the physiological changes inherent in the disease process. To do so, scientists must identify those virulence factors, or microbial gene products, that are specifically required for each stage of the infection process. Environmental conditions within the host are responsible for regulating the expression of most known virulence factors, (J. Mekalanos, J. Bacteriol. 174, 1 (1992)). Consequently, scientists attempt to mimic, in vitro, the environmental conditions within the host in an attempt to identify those genes that encode and are responsible for producing virulence factors. As a result, the identification of many virulence factors has been dependent on, and limited by, the ability of researchers to mimic host environmental factors in the laboratory.
There have been some methods developed for identifying virulence genes of microorganisms involved in pathogenesis. For example, a method referred to as insertional mutagenesis has long been recognized as a technique to inactivate and identify genes. Insertional mutagenesis relies on the ability of short stretches of DNA, known as insertion sequences, to move from one location to another on a chromosome by way of nonreciprocal recombination. Insertion sequences are not homologous with the regions of the plasmid or the chromosome into which the insert. Therefore, independent mutational events may be generated by randomly inserting an insertion sequence into a gene, thereby, disrupting the expression of that gene. As each mutated gene represents a different case, the selection procedure utilized in successfully recovering insertional routants is critical. In vitro assays are designed to screen for insertional activation events, i.e., the turning "on" of a previously silent gene, or insertional inactivation events, i.e., the turning "off" of a previously expressed gene. For an example of the insertional mutagenesis method see Fields et al., Proc. Natl. Acad. Sci. USA 83, 5189-5193, 1986.
The second basic technique utilized in the screen. Essentially, a piece of DNA or gene from the organism of interest is spliced into either a plasmid or a lambda phage, referred to as the vehicle or vector, and the resulting chimeric molecule is used to transform or infect, respectively, a host cell. A determination is then made as to whether the piece of DNA or gene of interest is capable of conferring a specific phenotype to the host cell which it would not otherwise possess, but for the gene of interest. For example, R. Isberg et al., in a technical paper entitled "A Single Genetic Locus Encoded by Yersinia pseudotuberculosis Permits Invasion of Cultured Animal Cells by Escherichia coli K-12," Nature, 317, 262-264, 1985, discloses a cloning screen in which a cosmid clone bank similar to that of a lambda phage, is prepared from Y. pseudotuberculosis and introduced into a bacterial E. coli K-12 strain. The E. coli K-12 strain containing random sequences of DNA representing the entire genetic information for Y. pseudotuberculosis was pooled, grown in broth, i.e., a complete medium, and used to infect a monolayer of cultured HEp-2 cells, i.e., animal cells. The cultured animal cells were then cultured and tested to determine whether introducing DNA from Y. pseudotuberculosis to E. coli confers an invasive phenotype typical of Y. pseudotuberculosis to E. coli.
A third method discussed by A. Osbourn et al., entitled "Identification of plant induced genes of the bacterial pathogen Xanthomonas campestris pathovar campestris using a promoter-probe plasmid", EMBO J., 6, 23-28, 1987, discloses a promoter probe plasmid for use in identifying promoters that are induced in vivo. Random chromosomal DNA fragments are cloned into a site in front of a promoterless chloramphenicol acetyltransferase gene contained in a plasmid. Transconjugates were then produced by transferring the resulting library into Xanthomonas. Individual transconjugates are then introduced into chloramphenicol-treated seedlings to determine whether the transconjugate displays resistance to chloramphenicol in the plant and then on an agar plate.
The final method utilized in the identification of genes is referred to as a regulatory screen. S. Knapp et al., in his technical publication, entitled "Two Trans-Acting Regulatory Genes (vir and mod) Control Antigenic Modulation in Bordetella pertussis," J. Bacteriol 170, 5059-5066, 1988, discloses a method for identifying potential virulence genes based on their coordinate expression with other known virulence genes under defined laboratory conditions.
The above technical papers by Fields et al., R. Isbert et al., and S. Knapp et al., each disclose methods for identifying microorganismal genes; however, the selection procedures or in vitro assays utilized in each method depends upon the ability of the in vitro assay to mimic the environmental conditions within the host, i.e., the in vivo environmental conditions. A disadvantage of these approaches is that each requires some understanding of the environmental conditions necessary to obtain virulence expression. Consequently, scientists have resorted to mixing host cells with the pathogen of interest in vitro to approximate the host's environmental conditions. Short of an exact duplication of the host's environmental conditions, critical regulatory factors necessary for the expression of many virulence factors may be missing, thus making the identification of those genes responsible for encoding virulence factors impossible to identify.
While the technical paper by A. Osbourn, et al., discloses a method to screen for promoters that are induced in vivo, a disadvantage is that no feasible method exists to select genes of a particular class, that is, individual transconjugates must be screened one by one in individual seedlings to determine whether a promoter is inducible. A further disadvantage results from a phenomena referred to as a position effect. A. Osbourn et al., utilize an autonomous plasmid and therefore the regulation of the promoter may vary considerably from the regulation of the promoter as it is found in its natural environment on the Xanthomonas genome. Other complications that arise from the use of plasmids are copy number, stability and super coiling effects.
There is still a need, therefore, for a method or technique for identifying genes encoding virulence factors in their normal environment whose expression is regulated, or turned "on", by undetermined factors within the host.